Fungal cells are endowed with a sturdy cell wall that is essential for survival, because it protects the cell against lysis caused by the internal turgor pressure and against mechanical injury. Because the components of the wall and the enzymes responsible for their formation are not present in mammalian cells, the cell wall is an obvious target for antifungal agents. The wall also imparts shape to the cell and is a dynamic structure, because its synthesis must accompany cell growth. For these reasons, the cell wall of budding yeast, Saccharomyces cerevisiae, has been used in my laboratory for many years as a model for morphogenesis, a process that takes place in all living organisms. The yeast cell wall is composed of polysaccharides and mannoproteins, which are linked together to form a network that is very resistant to mechanical stress. Thus, as we have previously demonstrated, the main structural component, beta(1-3)glucan, is linked to beta(1- 6)glucan, to which mannoproteins are attached. Chitin, a minor component, which is however essential for viability, is found in part free and in part bound to both beta(1-3) and beta(1- 6)glucan (1). Most of the chitin, synthesized through Chs3, is found in a ring at the neck between mother and daughter cell. The remainder is scattered through the cell wall, except for a small amount, unlinked to glucan, that forms the primary septum. The primary septum synthesis is catalyzed by Chs2. I have studied the linkage of chitin to different components of the cell wall and its function in the control of morphogenesis. These studies have been carried out in collaboration with Javier Arroyos laboratory at the Complutense University of Madrid, Spain, and with Vladimir Farkass laboratory at the Slovak Academy of Sciences, Bratislava, Slovakia. The starting point for these investigations was our finding that in cla4 mutants, partially compromised in function of the septin ring, inhibition of synthesis of the chitin ring resulted in growth at the mother-bud neck, followed by bud elongation, failure of cytokinesis and death of the cell (2). Normally, the mother-bud neck does not change in diameter after bud emergence and is the site of cytokinesis and cell separation. The septin ring, found inside the plasma membrane, is essential for cytokinesis. These results led to the conclusion that the chitin and the septin rings redundantly control growth at the neck. When both are defective, growth at the neck ensues and cytokinesis is disrupted. It is probable that septins prevent growth at the neck because of their barrier function, by impeding access at that location of membrane proteins necessary for cell wall synthesis. As for the chitin ring, for which no function had been previously found, we conceived a hypothesis based on chemical linkages in the cell wall. As mentioned above, chitin is linked to both beta(1-3) and beta(1- 6)glucan. The non-reducing glucose residues at the end of beta(1-3)glucan to which chitin is attached are the same to which in other molecules beta(1-6)glucan is bound. We hypothesized that at the neck, where the chitin content is very high, this polysaccharide may prevalently bind to beta(1-3)glucan, thus competing out the beta(1-6)glucan and preventing also attachment of the beta(1-6)glucan-bound mannoproteins. If this linkage of chitin to beta(1-3)glucan also prevents rearrangements of the glucan necessary for cell wall synthesis, the formation of cell wall at the neck would be essentially blocked. This hypothesis makes three predictions: a) that most of the chitin at the neck would be bound to beta(1-3)glucan, whereas the chitin spread out on the lateral walls would be largely bound to beta(1-6)glucan; b) that the beta(1-3)glucan at the neck would be somewhat different from the remainder, to account for its lack of metabolism; c) that mere presence of the chitin ring would not be able to control growth at the neck, unless the chitin was bound to beta(1-3)glucan. The first prediction was validated by the finding that most of bound chitin was attached to beta(1-3)glucan at the neck and to &#946;(1-6)glucan in lateral walls (3). For the second prediction, beta(1-3)glucan, both free and bound to chitin, was isolated by a new mild procedure, solubilized by carboxymethylation and fractionated by size exclusion chromatography. Two fractions of beta(1-3)glucan were separated: one had a very wide size distribution and presumably represented the polysaccharide undergoing remodeling during growth; the other one was of extremely high molecular weight and appeared to be finished structural material. The beta(1-3)glucan bound to chitin was also of high molecular weight and therefore corresponded to quiescent material. Thus, it appears that attachment of chitin does interfere with remodeling of beta(1-3)glucan, as predicted by our hypothesis. This work, that was completed during the present fiscal year, also revealed the presence of a noncovalent complex between chitin and beta(1-3)glucan. A paper dealing with this study was recently published and highlighted by the journal (4). To verify the third prediction, that chitin must be linked to beta(1-3)glucan to control growth, it was necessary to establish how the linkage between chitin and glucan is created. In a series of studies we showed that two proteins, Crh1 and Crh2, act as transglycosylases, transferring fragments of chitin chains to both beta(1-3) and beta(1- 6)glucan (5, 6, 7). In their absence, no chitin is bound to glucan. Thus, disruption of the CRH1 and CRH2 genes in a cla4&#916; strain allowed us to observe the effect of eliminating chitin linkages to glucan, without affecting chitin itself, in cells compromised in septin function. The triple mutant exhibited dramatic morphological aberrations, including wide necks, elongated buds and bloated cells. Despite containing chitin rings and four times the chitin amount of wild type, its appearance was practically identical to that of a cla4delta chs3delta double mutant, that was devoid of chitin, except for the primary septum. Note that although deletion of CHS3 and CLA4 should have led to synthetic lethality, in this case we were able to isolate the double mutant, in a different genetic background from that used in the initial genetic screen. By deleting the SWE1 gene in the triple mutant crh1delta crh2delta cla4delta, we eliminated the morphogenetic checkpoint and abolished bud elongation, but not neck widening. These results clearly show that chemical linkage of chitin to beta(1-3)glucan is necessary for control of growth at the mother-bud neck and define a new paradigm, where chemistry controls morphogenesis. This last study was mainly carried out in previous periods, but was completed in the present one. A paper on this work has been submitted. 1. Cabib, E., Roh, D.-H., Schmidt, M., Crotti, L.B., and Varma, A. (2001) J. Biol. lChem. 276, 19679-19682. 2. Schmidt, M., Varma, A., Drgon, T., Bowers, B., and Cabib, E. (2003) Mol. Biol. Cell 14, 2128-2141. 3. Cabib, E. and Durn, A. (2005) J. Biol. Chem. 280, 9170-9179. 4. Cabib, E., Blanco, N., and Arroyo, J. (2012) Eukaryot. Cell 11, 388-400. 5. Cabib, E., Blanco, N., Grau, C., Rodrguez-Pea, J.M., and Arroyo, J. (2007) Mol. Microbiol. 63, 921-935. 6. Cabib, E., Farkas, V., Kosk, O., Blanco, N., Arroyo, J., and McPhie, P. (2008) J. Biol. Chem. 283, 29859-29872. 7. Cabib, E. (2009) Eukaryot. Cell 8, 1626-1636.